Even with the successful creation of a transgenic, nuclear transfer, or cloned embryo, we are still faced with equal odds of developing female and male offspring. Recent investigations have suggested that multiple-photon excitation fluorescence microscopy (MPEFM) is a minimally invasive technique for observing the developing embryo. Furthermore, sex-specific markers such glucose-6-phosphate dehydrogenase (G6PDH) levels and H-Y antigen expression can predict embryo sex. Genetic sex can be confirmed using PCR, but the technique is invasive and costly. The overall goal of this project is to develop an assay which would allow for the rapid sexing of research embryos, with the eventual application to the commercial dairy and beef industries. Our approach for assessing the success of this assay will come through investigations evaluating 1) the development of bovine embryos imaged with a novel UPEFM, 2) the detection of the X-linked G6PDH message using antisense technology by NTEFM, 3) the detection of H-Y antigen on developing morulae and blastocysts using MPEFM, and 4) the success of combining G6PDH cytosolic levels and H-Y antigen detection to reliably predict genetic sex. The information obtained during these experiments will add to our fundamental knowledge of embryo development using real-time microscopy. The development of an assay to correctly predict the sex of a genetically manipulated embryo prior to transfer will increase the rate at which these advances are realized. Furthermore, this research will have direct application in improving the reproductive efficiency in domestic species.